The present invention relates to substances and their therapeutic use, and in particular to specific regions of p21WAF1 that bind to G1 and S phase specific cyclins, preferably ones activating CDK2 and to substances and mimetics based on this region. The invention also relates to assay methods and means for identifying substances useful for interfering with protein-protein interactions involving cyclins, particularly CDK/cyclin interactions and preferably capable of inhibiting CDK2 activity.
p21WAF1 is an inhibitor of both the G1 cyclin dependent protein kinases (CDKs; which control the progression from G1 into S phase) (Harper et al., 1995) and proliferating cell nuclear antigen (PCNA; an essential DNA-replication factor) (Florez-Rozas et al., 1994; Waga et al., 1994). Thus, inhibition of the function of either CDKs or PCNA provides, in theory, two distinct avenues for drug discovery based on the activity of p21WAF1. The PCNA binding function of p21WAF1 can be mimicked by a 20-amino acid peptide derived from the C-terminal domain of p21WAF1 and this peptide is sufficient partially to inhibit SV40 replication in vitro (Warbrick et al., 1995).
Despite its PCNA binding role, the primary function of the p21WAF1 protein as a growth suppressor appears to be inhibition of the G1 cyclin-CDK complexes (Chen et al., 1995; Harper et al., 1995; Luo et al., 1995; Nakanishi et al., 1995b). Luo et al. (1995) reported the N-terminal domain of p21 composed of residues 1-75, to act as a CDK-inhibitor in vitro, inhibiting cyclin E-CDK2.
WO 97/42222 (Cyclacel Ltd) discloses peptide fragments of p21WAF1 that interact with CDK4/cyclin D1. Thus it was observed that p21(16-35) and p21(46-65) bind to CDK4 and cyclin D1 respectively. Of these, only p21(16-35) was observed to inhibit CDK activity. p21(141-160) was observed to bind to CDK4 and cyclin D1 and to be a potent inhibitor of CDK4.
This data supported the known phenomenon of peptides including the sequence LFG as being the binding motif essential for the interaction of the p21 family with cyclins [Chen J et al.(1996), Lin J et al. and Russo A A et al.] and the further known properties of the amino-terminal half of p21 as being required for binding to CDK complex.
It should be borne in mind when considering the prior art discussed herein that unless otherwise explicitly stated the references to “motifs” is made with reference to papers that have made deductions and predictions based upon the activity of longer peptides usually consisting of at least 12 amino acids. Thus, the motifs are no more than conjecture based upon the a specific set of reactions. Such motifs provide no indication as to the actual length of peptide or modifications that could be made to retain and/or even enhance activity or specificity.
The sequence p21(141-160) (disclosed in WO97/42222 and Ball K. et al) in respect of cyclin D1/CDK4 inhibition was subjected to analysis in order to determine the minimum length of an inhibitory peptide upon which novel antiproliferative drugs could be designed. Observations of CDK4/cyclin D1 inhibitory activity led to the identification of an inhibitory motif comprising RRLIF (p21(155-159)) (SEQ ID No. 5), the bold residues being described as essential for activity and the underlined residue contributing towards inhibitory activity. Further observations in these disclosures include the retention of inhibitory activity against cyclin D1-CDK4 by the peptide KRRLIFSK (p21(154-161)) (SEQ ID No. 6) albeit at a concentration 1000 times greater than the parent sequence p21141-160 and that the substitution of aspartic acid at position 149 of p21141-160 by alanine surprisingly reduced the IC50 of the full length peptide from 100 nM to 46 nM. Thus, although identifying the RRLIF (SEQ ID No. 5) motif as being important to cyclinD1/CDK4 inhibition, Ball et al. is inconclusive as to the actual minimum length peptide required for enhanced activity. The effect of the Asp149 to Ala substitution has not proven reproducible.
In summary, WO97/42222 and Ball et al teach that there are sequences within the carboxy terminal region of p21 that are capable of interacting with CDK4/cyclin D in a manner that is inhibitory to CDK4 and further involves specific binding to cyclin D. Though the peptide p21(141-160) is described as being preferred, an 8-mer comprising p21(154-161) (KRRLIFSK) (SEQ ID No. 6) was inhibitory, but at higher concentrations. Finally, alanine replacement at position 149 within p21141-160 increased the inhibitory activity.
Thus, although the art indicates that this is an interesting region of p21 to investigate, no guidance is provided as to the identity of further fragments that would be preferably active against CDK4/cyclin D or any other CDK/cyclin enzymes.
Chen J et al. (Mol Cell Biol (1996) 16(9) 4673-4682) disclose a 12-mer corresponding to p2117-24 as being a cyclin binding domain of p21. They further identify a less avid cyclin binding region as p21150-161. Mutation and inhibition analysis demonstrated that the principal site of interaction with cyclin A was p2117-24, being a better inhibitor than p21150-161 consistent with its greater avidity for cyclins such that it can be detected by pull-down assay. Interaction of p21150-161 could only “be inferred from competition for binding and kinase inhibition assays. The importance of the p21150-161 in vivo was questioned due to the possibility of the relevant site being occupied by PCNA.
Adams D A et al. (Mol Cell Biol (1996) 16(12) 6623-6633) discloses N- and C-terminal regions of p21 that putatively bind to CDK2/cyclin. A 14-mer (p21149-162) is disclosed as inhibiting the binding of cyclin A to E2F1 and the binding of cyclins A and E to GST-p21. An amino acid sequence containing 8 amino acid residues (PVKRRLDL) (SEQ ID No. 7), derived from the transcription factor E2F1 was shown to bind to cyclin A/E-CDK2 complexes. An alanine scan of the 8-mer identified, on a qualitative level that certain modified forms of the peptide retained this activity. Noteworthy is that deletion or alanine replacement of either terminal amino acid reduced or abolished the ability to compete with GST-E2F1 for cyclin A binding.
In a further paper, Adams D A et al. (Mol Cell Biol (1999) 19(2) 1068-1080) investigated the existence of an E2F1-like motif within pRB as a means to explain its interaction with cyclin A/CDK2. A single 10-mer, pRB869-878 was the shortest pRB derived peptide investigated.
In a subsequent paper, Chen et al. (Proc. Natn. Acad. Sci. (1999) 96, 4325-4329) disclosed two E2F1 derived 8-mers as possessing the ability to interact with the cyclin A/CDK2 complex, being PVKRRLFG (SEQ ID No. 8) and PVKRRLDL (SEQ ID No. 7). These peptides were tested in whole cell assays using membrane translocation carrier peptides HIV-TAT or Penetratin®.
Brown N R et al. (Nature Cell Biol. (1999) 1, 438-443) describe a crystal structure of the cyclin A3/phospho-CDK2 complex with an 11-mer derived from p107 including the RXLF SEQ ID No. 9) motif. Of the 11-mer, the region RRLFGE (SEQ ID No. 10), was found to be within the binding region of cyclin A forming interactions with M210, I213, W217, E220, L253 and Q254.
An aim of the present invention has been to identify further peptides derived from p21 that retain or improve upon the inhibitory activities described in the art, particularly with regard to substrate specificity and peptide chain length as described in detail below.